CRH and POMC Effects on Animal Growth

ABSTRACT

The present invention provides for selection of bovine animals that will display phenotypes associated with increased rates of growth. These phenotypes include hot carcass weight, average daily gain, shipping weight, end of test rib eye area, and adjusted weaning weight which is a measure of post-natal growth, based on the knowledge of their CRH, POMC and MC4R genotypes. The predictive value comes from the discovery that certain single nucleotide polymorphisms (SNPs) in these genes are linked to higher growth rate phenotypes. Specifically, the phenotypes that correlated with specific SNP&#39;s are end-of-test rib-eye area, adjusted weaning weight, average daily gain, shipping weight and hot carcass weight. The invention also provides novel kits that can be used in making the determination of these genotypes. The invention further provides for methods of screening bovines to predict which animals will have higher growth rate, allowing producers to selectively breed and manage animus based on desired characteristics, thereby maximizing productivity and profitability in commercial meat production operations.

This application is a divisional patent application of U.S. Ser. No.11/790,511 filed on Apr. 26, 2007, which is a divisional patentapplication of U.S. Ser. No. 10/814,760 filed on Mar. 31, 2004.

The present invention relates generally to methods of selective breedingand management of livestock animals based on particular allelicpolymorphisms, and particularly to predicting the growth characteristicsof livestock animals based on these allelic polymorphisms.

BACKGROUND OF THE INVENTION

Increased growth rates are typically associated with higher economicreturns to beef producers. Consequently, methods to improve growth ratein cattle are of significant benefit to producers. Prior art methods ofincreasing growth has included such approaches as the use of hormoneimplants (Bagley et al., 1989), sub-therapeutic levels of antibiotics(Schumann et al., 1990) and by selective breeding based on expectedprogeny differences (EPD) (Kress et al., 1977). However, hormoneimplants and the use of antibiotics are becoming unpopular and may bebanned in North America in the near future. Therefore, alternativemethods of improving growth rates of cattle that don't requireartificial forms of stimulation will become increasingly important anddesirable in the industry.

Corticotropin releasing hormone (CRH) indirectly causes the release ofglucocorticoids (Dunn and Berridge, 1990), naturally occurring hormonesthat are suggested to be growth inhibitors (Sharpe et al., 1986).Although commonly referred to as a “stress-related hormone”, CRH isreleased from the hypothalamus, an area of the brain known to beinvolved in appetite control. The release of CRH regulates appetite viatwo distinct mechanisms: (1) indirectly triggering the release ofpro-opiomelanocortin (POMC), and (2) by increasing the production ofleptin (FIG. 1)

The up-regulation of POMC levels leads to increased synthesis of alphamelanocyte stimulating hormone (αMSH) which, when bound to themelanocortin-4 receptor, reduces appetite (Marsh et al., 1999). Theincrease of leptin, which is induced by glucocorticoids, reducesappetite by four other interactions (FIG. 1). Primarily leptin acts todecrease the levels of neuropeptide Y, an appetite stimulant. Leptinalso acts to increase POMC levels, an agonist for the melanocortin4-receptor (MC4R); decrease the levels of antagonist agouti relatedprotein (AGRP); and increase the production of CRH (Houseknecht et al.,1998; Marsh et al., 1999; Pritchard et al., 2002).

The CRH gene comprises two exons, however only exon 2 is translated andcodes for the pre-pro-protein (Roche et al., 1988; Shibihara et al.,1983). The CRH gene has been mapped to chromosome 14 (Barendse et al.,1997), and the results of quantitative trait linkage (QTL) mappingsuggested an association between a locus for post-natal growthidentified on chromosome 14 and the CRH gene (Buchanan et al., 2000). Inaddition, we previously reported a non-conserved amino acid substitutionat position 77 (CRH77) in the pro-peptide region of CRM and showed anassociation with post-natal growth in beef cattle (Buchanan et al.,2002b).

The POMC pro-hormone peptide is an integral component of the appetiteregulation pathway (FIG. 1) and has also been identified by QTL analysisin our unpublished studies as a positional candidate gene for averagedaily gain and carcass weight. We identified a single nucleotidepolymorphism (SNP) in the POMC gene that is translationally silent andused the SNP to map the POMC gene to chromosome 11 in beef cattle (Thueet al., 2003), confirming its position to previously identified QTLloci. We also identified SNPs in two other genes integral to thispathway, leptin and MC4R (Buchanan et al., 2002a; Thue et al., 2001).

We have recently identified a novel SNP in the CRH gene, at position 4of the signal sequence, equivalent to position 22 of the sequencedefined in SEQ ID NO: 1. Together with the existing gene tests for POMC,MC4R and LEP (Buchanan et al., 2002a; Buchanan et al., 2002b; Thue etal., 2001; Thue et al., 2003) we genotyped a group of 256 steers. Ourresults show that knowledge of genotypes of cattle, with respect tothese particular genes, can be used to better predict growth and yieldduring beef production.

SUMMARY OF THE INVENTION

It is well known to those skilled in the art that single nucleotidepolymorphisms (SNPs) can provide a useful way in which to distinguishdifferent alleles of a gene. Furthermore, when the presence of a SNP canbe associated with a specific phenotype, the SNP operates as a powerfulmarker and can be used to predict phenotypic outcomes based on ananimal's genotypic makeup. The present invention relates to methods ofmanaging livestock animals, such as cattle and pigs, and takingadvantage of genetic factors that affect an animal's appetite. Byidentifying animals with a particular genotype, with respect to hereindescribed SNP alleles, it is possible to identify animals that willdisplay phenotypes associated with increased growth rate, as compared toanimals lacking the desired genotype.

In particular, the present invention relates to methods for establishingthe genetically determined predispositions of individual livestockanimals, such as cattle and pigs, within a group of such animals, tomeet particular desired characteristics with respect to growth, based onthe association of specific CRH, POMC or MC4R alleles with an increasedappetite and hence growth phenotype.

The present invention provides a method for analyzing the genotype ofanimals with respect to the CRH, POMC and MC4R genes, and using thegenotype information to select animals with desired traits related toanimal growth. Such knowledge further permits producers to charge apremium for the more desirable faster-growing phenotype, and permitsbreeders to selectively breed animals for genotypes that will result inthe most desirable phenotypes.

It is therefore an object of the present invention to provide a methodfor selecting for animals homozygous for the “G” allele at the CRH genelocus, in the knowledge that animals that are “GG” homozygotes willdisplay the desired phenotypes of increased hot carcass weight,increased end of test rib eye area, and increased adjusted weaningweight.

It is a further object of the present invention to provide a method forselecting for animals homozygous for the “T” allele at the POMC genelocus, in the knowledge that animals that are “TT” homozygotes willdisplay the desired phenotypes of increased average daily gain, shippingweight and hot carcass weight.

It is still another object of the present invention to provide a methodfor selecting for animals having at least one “C” allele at the MC4Rgene locus, in the knowledge that animals that are “CG” or “CC” at theMC4R gene locus will display the desired phenotype of increase hotcarcass weight.

It is a further object of the present invention to select for animalshomozygous for the “G” allele at CRH and the “T” allele at POMC loci, inthe knowledge that those animals that are “GG-TT” homozygotes willdisplay concurrent increases in rib-eye area, shipping weight, hotcarcass weight and average daily gain.

It is a further object of the present invention that where the desiredphenotype is only increased hot carcass weight, that a more efficientmethod of testing animals is provided, wherein an animal is first testedto determine it's MC4R genotype in the knowledge that an animal with atleast on “C” allele at MC4R will display the desired phenotype ofincreased carcass weight, regardless of the animal's CRH genotype, suchthat only those animals that are “GG” at MC4R will need to be testedwith respect to their CRH genotype in order to determine whether theywill display the desired phenotype of increased hot carcass weights.

It is a further object of the present invention to provide a diagnostickit to be used in the determination of an animal's CRH, POMC and MC4Rgenotype.

These and other objects, features, and advantages of the inventionbecome further apparent in the following detailed description of theinvention when taken in conjunction with the accompanying drawings whichillustrate, by way of example, the principles of this invention.

Thus, the invention provides a genetic testing method for thedetermination of an animal's CRH, POMC and MC4R genotype based onanalysis of the presence or absence of specific SNP's, and the use ofthe knowledge of an animal's genotype such that animals of like genotypecan be identified and selected according to the desired phenotypes ofincreased shipping weight, hot carcass weight, average daily gain andrib-eye area.

DESCRIPTION OF THE FIGURES

While the invention is claimed in the concluding portions hereof,preferred embodiments are provided in the accompanying detaileddescription which may be best understood in conjunction with theaccompanying diagrams where like parts in each of the several diagramsare labeled with like numbers, and where:

FIG. 1: Appetite pathway. Arrows show the effect of leptins and CRH onneuropeptides that control appetite. Ovals represent neurons in thehypothalamus. CRH—corticotrophin-releasing hormone, AGRP—agouti relatedprotein, POMC—pro-opiomelanocortin, NPY—neuropeptide Y, αMSH—alphamelanocyte stimulating hormone and MC4R—melanocortin 4 receptor.

FIG. 2: Sequence of the CRH SNP with respect to the nucleotide andprotein coding sequences. The SNP of the invention occurs at position 22of the nucleotide sequence as defined by SEQ ID NO: 1, which correspondsto codon 4 of the protein coding sequence.

FIG. 3: The effect of CRH genotype on CRH secretion.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In the description that follows, a number of terms used in recombinantDNA technology are extensively utilized. In order to provide a clear andconsistent understanding of the specification and claims, including thescope to be given such terms, the following definitions are provided:

By “amplifying a segment” as used herein, is meant the production ofsufficient multiple copies of the segment to permit relatively facilemanipulation of the segment. Manipulation refers to both physical andchemical manipulation, that is, the ability to move bulk quantities ofthe segment around and to conduct chemical reactions with the segmentthat result in detectable products.

A “segment” of a polynucleotide refers to an oligonucleotide that is apartial sequence of entire nucleotide sequence of the polynucleotide.

A “modified segment” refers to a segment in which one or more naturalnucleotides have been replaced with one or more modified nucleotides. A“modified, labeled segment” refers to a modified segment that alsocontains a nucleotide, which is different from the modified nucleotideor nucleotides therein, and which is detectably labeled.

An “amplification primer” is an oligonucleotide that is capable ofannealing adjacent to a target sequence and serving as an initiationpoint for DNA synthesis when placed under conditions in which synthesisof a primer extension product which is complementary to a nucleic acidstrand is initiated.

By “analysis” is meant either detection of variations in the nucleotidesequence among two or more related polynucleotides or, in thealternative, determining the full nucleotide sequence of apolynucleotide. By “analyzing” the hybridized fragments for anincorporated detectable label identifying the suspected polymorphism ismeant that, at some stage of the sequence of events that leads tohybridized fragments, a label is incorporated. The label may beincorporated at virtually any stage of the sequence of events includingthe amplification, cleavage or hybridization procedures. The label mayfurther be introduced into the sequence of events after cleavage andbefore or after hybridization. The label so incorporated is thenobserved visually or by instrumental means. The presence of the labelidentifies the polymorphism due to the fact that the fragments obtainedduring cleavage are specific to the modified nucleotide(s) used in theamplification and at least one of the modified nucleotide is selected soas to replace a nucleotide involved in the polymorphism.

The term “animal” is used herein to include all vertebrate animals,including humans. It also includes an individual animal in all stages ofdevelopment, including embryonic and fetal stages. As used herein, theterm “production animals” is used interchangeably with “livestockanimals” and refers generally to animals raised primarily for food. Forexample, such animals include, but are not limited to, cattle (bovine),sheep (ovine), pigs (porcine or swine), poultry (avian), and the like.As used herein, the term “cow” or “cattle” is used generally to refer toan animal of bovine origin of any age. Interchangeable terms include“bovine”, “calf”, “steer”, “bull”, “heifer” and the like. As usedherein, the term “pig” or is used generally to refer to an animal ofporcine origin of any age. Interchangeable terms include “piglet”, “sow”and the like.

The term “antisense” is intended to refer to polynucleotide moleculescomplementary to a portion of an RNA marker of a gene, as definedherein. “Complementary” polynucleotides are those that are capable ofbase pairing according to the standard Watson-Crick complementarityrules, where purines base pair with pyrimidines to form combinations ofguanine paired with cytosine (G:C) and adenine paired with eitherthymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) inthe case of RNA. Inclusion of less common bases like inosine,5-methylcytosine, 6-methyladenine, hypoxanthine and others inhybridizing sequences does not interfere with pairing.

By the term “complementarity” or “complementary” is meant, for thepurposes of the specification or claims, a sufficient number in theoligonucleotide of complementary base pairs in its sequence to interactspecifically (hybridize) with the target nucleic acid sequence of thegene polymorphism to be amplified or detected. As known to those skilledin the art, a very high degree of complementarity is needed forspecificity and sensitivity involving hybridization, although it neednot be 100%. Thus, for example, an oligonucleotide that is identical innucleotide sequence to an oligonucleotide disclosed herein, except forone base change or substitution, may function equivalently to thedisclosed oligonucleotides. A “complementary DNA” or “cDNA” geneincludes recombinant genes synthesized by reverse transcription ofmessenger RNA (“mRNA”).

By the term “composition” is meant, for the purposes of thespecification or claims, a combination of elements which may include oneor more of the following: the reaction buffer for the respective methodof enzymatic amplification, plus one or more oligonucleotides specificfor CRH, POMC or MC4R gene polymorphisms, wherein said oligonucleotideis labeled with a detectable moiety.

A “cyclic polymerase-mediated reaction” refers to a biochemical reactionin which a template molecule or a population of template molecules isperiodically and repeatedly copied to create a complementary templatemolecule or complementary template molecules, thereby increasing thenumber of the template molecules over time, The products of such areaction are commonly referred to as amplification products.

“Denaturation” of a template molecule refers to the unfolding or otheralteration of the structure of a template so as to make the templateaccessible for duplication or hybridization. In the case of DNA,“denaturation” refers to the separation of the two complementary strandsof the double helix, thereby creating two complementary, single strandedtemplate molecules. “Denaturation” can be accomplished in any of avariety of ways well known to those skilled in the art, including heator by treatment of the DNA with a base or other chemical denaturant.

A “detectable amount of product” refers to an amount of amplifiednucleic acid that can be detected using standard laboratory tools. A“detectable marker” refers to a nucleotide analog that allows detectionusing visual or other means. For example, fluorescently labelednucleotides can be incorporated into a nucleic acid during one or moresteps of a cyclic polymerase-mediated reaction, thereby allowing thedetection of the product of the reaction using, e.g. fluorescencemicroscopy or other fluorescence-detection instrumentation.

By the term “detectable moiety” is meant, for the purposes of thespecification or claims, a label molecule (isotopic or non-isotopic)which is incorporated indirectly or directly into an oligonucleotide,wherein the label molecule facilitates the detection of theoligonucleotide in which it is incorporated when the oligonucleotide ishybridized to amplified gene polymorphism sequences. Thus, “detectablemoiety” is used synonymously with “label molecule”. Synthesis ofoligonucleotides can be accomplished by any one of several methods knownto those skilled in the art Label molecules, known to those skilled inthe art as being useful for detection, include chemiluminescent orfluorescent molecules. Various fluorescent molecules are known in theart that are suitable for use to label a nucleic acid substrate for themethod of the present invention. The protocol for such incorporation mayvary depending upon the fluorescent molecule used. Such protocols areknown in the art for the respective fluorescent molecule.

By “detectably labeled” is meant that a fragment or an oligonucleotidecontains a nucleotide that is radioactive, that is substituted with afluorophore or some other molecular species that elicits a physical orchemical response can be observed by the naked eye or by means ofinstrumentation such as, without limitation, scintillation counters,colorimeters, UV spectrophotometers and the like. As used herein, a“label” or “tag” refers to a molecule that, when appended by, forexample, without limitation, covalent bonding or hybridization, toanother molecule, for example, also without limitation, a polynucleotideor polynucleotide fragment, provides or enhances a means of detectingthe other molecule. A fluorescence or fluorescent label or tag emitsdetectable light at a particular wavelength when excited at a differentwavelength. A radiolabel or radioactive tag emits radioactive particlesdetectable with an instrument such as, without limitation, ascintillation counter. Other signal generation detection methodsinclude: chemiluminescence, electrochemiluminescence, ramanspectroscopy,colorimetric, hybridization protection assay, and Mass spectrometry.

“DNA amplification” as used herein refers to any process that increasesthe number of copies of a specific DNA sequence by enzymaticallyamplifying the nucleic acid sequence. A variety of processes are known.One of the most commonly used is the polymerase chain reaction (PCR)process of Mullis as described in U.S. Pat. Nos. 4,683,195 and4,683,202. PCR involves the use of a thermostable DNA polymerase, knownsequences as primers, and heating cycles, which separate the replicatingdeoxyribonucleic acid (DNA), strands and exponentially amplify a gene ofinterest Any type of PCR, such as quantitative PCR, RT-PCR, hot startPCR, LA-PCR, multiplex PCR, touchdown PCR, etc., may be used.Preferably, real-time PCR is used. In general, the PCR amplificationprocess involves an enzymatic chain reaction for preparing exponentialquantities of a specific nucleic acid sequence. It requires a smallamount of a sequence to initiate the chain reaction and oligonucleotideprimers that will hybridize to the sequence. In PCR the primers areannealed to denatured nucleic acid followed by extension with aninducing agent (enzyme) and nucleotides. This results in newlysynthesized extension products. Since these newly synthesized productsbecome templates for the primers, repeated cycles of denaturing, primerannealing, and extension results in exponential accumulation of thespecific sequence being amplified. The extension product of the chainreaction will be a discrete nucleic acid duplex with a terminicorresponding to the ends of the specific primers employed.

“DNA” refers to the polymeric form of deoxyribonucleotides (adenine,guanine, thymine, or cytosine) in its either single stranded form, or adouble-stranded helix. This term refers only to the primary andsecondary structure of the molecule, and does not limit it to anyparticular tertiary forms. Thus, this term includes double-stranded DNAfound in linear and circular DNA molecules including, but not limitedto, restriction fragments, viruses, plasmids, cosmids, and artificialand naturally occurring chromosomes. In discussing the structure ofparticular double-stranded DNA molecules, sequences may be describedherein according to the normal convention of giving only the sequence inthe 5′ to 3′ direction along the non-transcribed strand of DNA (i.e.,the strand having a sequence homologous to the mRNA).

By the terms “enzymatically amplify” or “amplify” is meant, for thepurposes of the specification or claims, DNA amplification by anyprocess by which nucleic acid sequences are amplified in number. Thereare several means for enzymatically amplifying nucleic acid sequencesknown in the art. Currently the most commonly used method is thepolymerase chain reaction (PCR). Other amplification methods include LCR(ligase chain reaction) which utilizes DNA ligase, and a probeconsisting of two halves of a DNA segment that is complementary to thesequence of the DNA to be amplified, enzyme Qβ replicase and aribonucleic acid (RNA) sequence template attached to a probecomplementary to the DNA to be copied which is used to make a DNAtemplate for exponential production of complementary RNA; stranddisplacement amplification (SDA); Qβ replicase amplification (QβRA);self-sustained replication (3SR); and NASBA (nucleic acid sequence-basedamplification), which can be performed on RNA or DNA as the nucleic acidsequence to be amplified. The particular methodology used to amplify DNAsequences is not intended to be limiting, and all such it is intendedthat the scope of the invention will include all methods of DNAamplification known in the art.

The “extension of the primers” refers to the addition of nucleotides toa primer molecule so as to synthesize a nucleic acid complementary to atemplate molecule. “Extension of the primers” does not necessarily implythat the primer molecule is extended to synthesize a completecomplementary template molecule. Rather, even if only a fraction of thetemplate molecule has been copied, the primer is still consideredextended.

A “fragment” of a molecule such as a protein or nucleic acid is meant torefer to any portion of the amino acid or nucleotide genetic sequence.

By “heterozygous” or “heterozygous polymorphism” is meant that the twoalleles of a diploid cell or organism at a given locus are different,that is, that they have a different nucleotide exchanged for the samenucleotide at the same place in their sequences.

By “homozygous” is meant that the two alleles of a diploid cell ororganism at a given locus are identical, that is, that they have thesame nucleotide for nucleotide exchange at the same place in theirsequences.

By “hybridization” or “hybridizing,” as used herein, is meant theformation of A-T and C-G base pairs between the nucleotide sequence of afragment of a segment of a polynucleotide and a complementary nucleotidesequence of an oligonucleotide. By complementary is meant that at thelocus of each A, C, G or T (or U in the case of an RNA molecule) in thefragment sequence, the oligonucleotide sequenced has a T, G, C or A,respectively. The hybridized fragment/oligonucleotide is called a“duplex.” In the case of a DNA-RNA hybrid, the molecular is called a“heteroduplex.”

A “hybridization complex”, means a complex of nucleic acid moleculesincluding at least the target nucleic acid and sensor probe. It may alsoinclude an anchor probe.

By “immobilized on a solid support” is meant that a fragment, primer oroligonucleotide is attached to a substance at a particular location insuch a manner that the system containing the immobilized fragment,primer or oligonucleotide may be subjected to washing or other physicalor chemical manipulation without being dislodged from that location. Anumber of solid supports and means of immobilizing nucleotide-containingmolecules to them are known in the art; any of these supports and meansmay be used in the methods of this invention.

As used herein, the term “nucleic acid molecule” is intended to includeDNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA),analogs of the DNA or RNA generated using nucleotide analogs, andderivatives, fragments and homologs thereof. The nucleic acid moleculecan be single-stranded or double-stranded, but preferably isdouble-stranded DNA. An “isolated” nucleic acid molecule is one that isseparated from other nucleic acid molecules that are present in thenatural source of the nucleic acid. A “nucleoside” refers to a baselinked to a sugar. The base may be adenine (A), guanine (G) (or itssubstitute, inosine (I)), cytosine (C), or thymine (T) (or itssubstitute, uracil (U)). The sugar may be ribose (the sugar of a naturalnucleotide in RNA) or 2-deoxyribose (the sugar of a natural nucleotidein DNA). A “nucleotide” refers to a nucleoside linked to a singlephosphate group.

As used herein, the term “oligonucleotide” refers to a series of linkednucleotide residues, which oligonucleotide has a sufficient number ofnucleotide bases to be used in a PCR reaction. A short oligonucleotidesequence may be based on, or designed from, a genomic or cDNA sequenceand is used to amplify, confirm, or reveal the presence of an identical,similar or complementary DNA or RNA in a particular cell or tissue.Oligonucleotides may be chemically synthesized and may be used asprimers or probes. Oligonucleotide means any nucleotide of more than 3bases in length used to facilitate detection or identification of atarget nucleic acid, including probes and primers.

“Polymerase chain reaction” or “PCR” refers to a thermocyclic,polymerase-mediated, DNA amplification reaction. A PCR typicallyincludes template molecules, oligonucleotide primers complementary toeach strand of the template molecules, a thermostable DNA polymerase,and deoxyribonucleotides, and involves three distinct processes that aremultiply repeated to effect the amplification of the original nucleicacid. The three processes (denaturation, hybridization, and primerextension) are often performed at distinct temperatures, and in distincttemporal steps. In many embodiments, however, the hybridization andprimer extension processes can be performed concurrently. The nucleotidesample to be analyzed may be PCR amplification products provided usingthe rapid cycling techniques described in U.S. Pat. No. 5,455,175. Othermethods of amplification include, without limitation, NASBR, SDA, 3SR,TSA and rolling circle replication. It is understood that, in any methodfor producing a polynucleotide containing given modified nucleotides,one or several polymerases or amplification methods may be used. Theselection of optimal polymerization conditions depends on theapplication.

A “polymerase” is an enzyme that catalyzes the sequential addition ofmonomeric units to a polymeric chain, or links two or more monomericunits to initiate a polymeric chain. In preferred embodiments of thisinvention, the “polymerase” will work by adding monomeric units whoseidentity is determined by and which is complementary to a templatemolecule of a specific sequence. For example, DNA polymerases such asDNA Pol I and Taq polymerase add deoxyribonucleotides to the 3′ end of apolynucleotide chain in a template-dependent manner, therebysynthesizing a nucleic acid that is complementary to the templatemolecule. Polymerases may be used either to extend a primer once orrepetitively or to amplify a polynucleotide by repetitive priming of twocomplementary strands using two primers.

A “polynucleotide” refers to a linear chain of nucleotides connected bya phosphodiester linkage between the 3′-hydroxyl group of one nucleosideand the 5′-hydroxyl group of a second nucleoside, which in turn islinked through its 3′-hydroxyl group to the 5′-hydroxyl group of a thirdnucleoside and so on to form a polymer comprised of nucleosides liked bya phosphodiester backbone. A “modified polynucleotide” refers to apolynucleotide in which one or more natural nucleotides have beenpartially or substantially completely replaced with modifiednucleotides.

A “primer” is a short oligonucleotide, the sequence of which iscomplementary to a segment of the template which is being replicated,and which the polymerase uses as the starting point for the replicationprocess. By “complementary” is meant that the nucleotide sequence of aprimer is such that the primer can form a stable hydrogen bond complexwith the template; i.e., the primer can hybridize to the template byvirtue of the formation of base-pairs over a length of at least tenconsecutive base pairs.

The primers herein are selected to be “substantially” complementary todifferent strands of a particular target DNA sequence. This means thatthe primers must be sufficiently complementary to hybridize with theirrespective strands. Therefore, the primer sequence need not reflect theexact sequence of the template. For example, a non-complementarynucleotide fragment may be attached to the 5′ end of the primer, withthe remainder of the primer sequence being complementary to the strand.Alternatively, non-complementary bases or longer sequences can beinterspersed into the primer, provided that the primer sequence hassufficient complementarity with the sequence of the strand to hybridizetherewith and thereby form the template for the synthesis of theextension product.

A “restriction enzyme” refers to an endonuclease (an enzyme that cleavesphosphodiester bonds within a polynucleotide chain) that cleaves DNA inresponse to a recognition site on the DNA. The recognition site(restriction site) consists of a specific sequence of nucleotidestypically about 4-8 nucleotides long.

A “single nucleotide polymorphism” or “SNP” refers to polynucleotidethat differs from another polynucleotide by a single nucleotideexchange. For example, without limitation, exchanging one A for one C, Gor T in the entire sequence of polynucleotide constitutes a SNP. Ofcourse, it is possible to have more than one SNP in a particularpolynucleotide. For example, at one locus in a polynucleotide, a C maybe exchanged for a T, at another locus a G may be exchanged for an A andso on. When referring to SNPs, the polynucleotide is most often DNA andthe SNP is one that usually results in a change in the genotype that isassociated with a corresponding change in phenotype of the organism inwhich the SNP occurs.

As used herein, a “template” refers to a target polynucleotide strand,for example, without limitation, an unmodified naturally-occurring DNAstrand, which a polymerase uses as a means of recognizing whichnucleotide it should next incorporate into a growing strand topolymerize the complement of the naturally-occurring strand. Such DNAstrand may be single-stranded or it may be part of a double-stranded DNAtemplate. In applications of the present invention requiring repeatedcycles of polymerization, e.g., the polymerase chain reaction (PCR), thetemplate strand itself may become modified by incorporation of modifiednucleotides, yet still serve as a template for a polymerase tosynthesize additional polynucleotides.

A “thermocyclic reaction” is a multi-step reaction wherein at least twosteps are accomplished by changing the temperature of the reaction.

A “thermostable polymerase” refers to a DNA or RNA polymerase enzymethat can withstand extremely high temperatures, such as thoseapproaching 100° C. Thermostable polymerases are typically isolated fromorganisms that live in extreme temperatures, such as Thermus aquaticus.Examples of thermostable polymerases include Taq, Tth, Pfu, Vent, deepvent, UITma, and variations and derivatives thereof.

A “variant” is a difference in the nucleotide sequence among relatedpolynucleotides. The difference may be the deletion of one or morenucleotides from the sequence of one polynucleotide compared to thesequence of a related polynucleotide, the addition of one or morenucleotides or the substitution of one nucleotide for another. The terms“mutation,” “polymorphism” and “variant” are used interchangeably hereinto describe such variants. As used herein, the term “variant” in thesingular is to be construed to include multiple variances; i.e., two ormore nucleotide additions, deletions and/or substitutions in the samepolynucleotide. A “point mutation” refers to a single substitution ofone nucleotide for another.

The present invention makes use of a number of oligonucleotide sequencesas described herein as primers for use in DNA amplification reaction oras hybridization probes. It is well known to those skilled in the artthat such oligonucleotides may be modified in terms of length and evenspecificity, and they will still be well suited to the practice of theinvention. As such, the invention is not limited to the preciseoligonucleotides described but is intended to include all thoseoligonucleotides that will allow the method of the invention to becarried out as disclosed herein.

Introduction:

A variety of characteristics of livestock animals are consideredimportant in determining the overall value of the finished product. Somefactors are involved in the palatability of the meat produced, which isimportant to consumers, and which is reflected in the grading systemused to classify meat. Still other factors affect the cost of producingan animal of given size and therefore affect the cost of meat that theconsumer will ultimately pay, and which will result in improvedprofitability for producers of livestock as well as the operators offeedlots. As a result, methods of production that can improve thequality, or reduce the cost of production are desirable for allconcerned in the production and consumption of meat from livestock.

The present invention discloses the discovery of SNPs that areassociated with a variety of parameters related to the growth rate ofanimals. Knowledge of the CRH, POMC and MC4R genotype of animals permitsthe development of genetic testing methods such that animals with themost desirable characteristics with regard to carcass weight, averagedaily weight gain and rib eye area can be identified and selected. Thisin turn leads to the development of methods of livestock management,wherein a higher degree of predictability about the eventual developmentof livestock animals becomes possible, once the genotype of animals withregard to the CRH, POMC and MC4R genes is determined.

According to one aspect of the present invention, there is provided amethod for distinguishing bovines having a CRH gene polymorphism. Themethod comprises the steps of first isolating a genomic DNA sample froma bovine, and then amplifying a region of the bovine CRH gene using anoligonucleotide pair to form nucleic acid amplification products of CRHgene polymorphism sequences. Amplification can be by any of a number ofmethods known to those skilled in the art including PCR, and theinvention is intended to encompass any suitable methods of DNAamplification.

The amplification products are then analyzed in order to detect thepresence or absence of at polymorphism in the CRH gene that alters thefourth amino acid and which is associated with a number of desiredphenotypes. The polymorphism comprises a C to G transition at a positioncorresponding to position 22 of SEQ ID NO: 1, which is the cDNA sequenceof exon 2 of the Bos taurus corticotrophin-releasing hormone precursor(CRH) gene (GenBank Accession No. AF340152). The presence of a “G”residue at this position results in animals that display the desirablephenotypes of increased carcass weight, increased end-of-test rib-eyearea and increased post-natal growth. By practicing the method of thepresent invention and analyzing the amplification products it ispossible to determine the genotype of individual animals with respect tothe polymorphism.

Conveniently, analysis may be made by restriction fragment lengthpolymorphism (RFLP) analysis of a 129 bp PCR produced by amplificationof bovine genomic DNA with the oligonucleotide pair of SEQ ID NO: 4 andSEQ ID NO: 5. The use of a forward primer containing a purposefulmismatch, in combination with a target DNA containing the “G” allele,creates a DdeI site such that the presence of the “G” allele ispositively indicated by digestion of the amplification products withDdeI. In the absence of the “G” allele, the amplification product stillcontains a single DdeI site, normally present in the CRH gene, such thatdigestion yields two fragment of 88 and 41 bp. In the presence of theSNP, the “G” allele, an additional DdeI site is created at nucleotide 87of the amplification product, resulting in the further digestion of the88 bp fragment to yield fragments of 69 and 19 bp.

In order to simplify detection of the amplification products and therestriction fragments, it will be obvious to those skilled in the artthat the amplified DNA will further comprise labeled moieties to permitdetection of relatively small amounts of product. A variety of moietiesare well known to those skilled in the art and include such labelingtags as fluorescent, bioluminescent, chemiluminescent, and radioactiveor colorigenic moieties.

A variety of methods of detecting the presence and restriction digestionproperties of CRH gene amplification products are also suitable for usewith the present invention. These can include methods such as gelelectrophoresis, mass spectroscopy or the like. The present invention isalso adapted to the use of single stranded DNA detection techniques suchas fluorescence resonance energy transfer (FRET). For FRET analysis,hybridization anchor and detection probes may be used to hybridize tothe amplification products. The probes sequences are selected such thatin the presence of the SNP (i.e. a G residue at position 22 as describedabove), the resulting hybridization complex is more stable than if thereis a C residue at the same position. By adjusting the hybridizationconditions, it is therefore possible to distinguish between animals withthe SNP and those without. A variety of parameters well known to thoseskilled in the art can be used to affect the ability of a hybridizationcomplex to form. These include changes in temperature, ionicconcentration, or the inclusion of chemical constituents like formamidethat decrease complex stability. It is further possible to distinguishanimals heterozygous for the SNP versus those that are homozygous forthe same. The method of FRET analysis is well known to the art, and theconditions under which the presence or absence of the SNP would bedetected by FRET are readily determinable.

It is also well known to those in the art that a number of DNAamplification techniques are suitable for use with the presentinvention. Conveniently such amplification techniques may comprisemethods such as polymerase chain reaction (PCR), strand displacementamplification (SDA), nucleic acid sequence based amplification (NASBA),rolling circle amplification, T7 polymerase mediated amplification, T3polymerase mediated amplification and SP6 polymerase mediatedamplification. The precise method of DNA amplification is not intendedto be limiting, and other methods not listed here will be apparent tothose skilled in the art and their use is within the scope of theinvention.

In another aspect, the present invention provides a method fordistinguishing bovines having a POMC gene polymorphism. The methodcomprises isolating genomic DNA from a bovine, amplifying a region ofthe bovine POMC gene using an oligonucleotide pair to form nucleic acidamplification sequences comprising amplified POMC gene polymorphismsequences, and then analyzing the amplification products in order todetect the presence or absence of a SNP in the POMC gene at position 254of SEQ ID NO: 2. The polymorphism is a C to T transition, such that thepresence of a “T” residue at this is position is associated with thedesirable phenotypes of increased shipping weight and increased averagedaily gain, as compared to animals with a “C” residue at position 254 ofSEQ ID NO: 2.

Conveniently, the POMC SNP can be detected by restriction digest of a390 bp amplification product produced using the oligonucleotide pair SEQID NO: 6 and SEQ ID NO: 7. The presence of a “T” residue creates a BtsIrestriction site at nucleotide 157 in a nucleic acid amplificationproduct produced by amplification of the POMC gene using theoligonucleotide pair SEQ ID NO: 6 and SEQ ID NO: 7. The presence of theSNP is readily detected by digestion of the amplification product withBtsI, and the digestion products analyzed by methods such as gelelectrophoresis and the like.

As was described above, in order to simplify detection of theamplification products and the restriction fragments, it will be obviousto those skilled in the art that the amplified DNA will further compriselabeled moieties to permit detection of relatively small amounts ofproduct.

In practicing the present invention it is also possible to use otherknown methods of analysis, such as FRET analysis, as a method ofdetection. Conveniently, hybridization probes comprising an anchor anddetection probe, the design of which art is well known to those skilledin the art of FRET analysis, are labeled with a detectable moiety, andthen under suitable conditions are hybridized a POMC amplificationproduct containing the site of interest in order to form a hybridizationcomplex. Specifically, the hybridization probe will be designed suchthat a change at position 254 of SEQ ID NO: 2 will produce ahybridization complex of altered stability. A variety of parameters wellknown to those skilled in the art can be used to affect the ability of ahybridization complex to form. These include changes in temperature,ionic concentration, or the inclusion of chemical constituents likeformamide that decrease complex stability.

The presence or absence of the POMC SNP is then determined by thestability of the hybridization complex as was described for the CRHgene. The parameters affecting hybridization and FRET analysis are wellknown to those skilled in the art. In addition, the foregoing areexamples of amplification products and hybridization probes that aresuitable for use with FRET analysis. It will be readily apparent tothose skilled in the art that modification may be made to theoligonucleotides that are used to synthesize the amplification productsor probes while still permitting the practice of the present invention.As before, a variety of amplification methods are suitable for use inthe practice of the present invention and all such methods are intendedto be within the scope of the invention.

In another aspect, the present invention provides a method fordistinguishing animals having a MC4R gene polymorphism. The methodcomprises isolating genomic DNA from a bovine, amplifying a region ofthe bovine MC4R gene using an oligonucleotide pair to form nucleic acidamplification sequences comprising amplified MC4R gene polymorphismsequences, and then detecting a SNP present in the MC4R gene. The SNPcomprises a G to C transition at position 1069 of SEQ ID NO: 3, and isassociated with the phenotype of maximum increased hot carcass weight,as compared to bovines with a “G” residue at this position.

Conveniently, a portion of the MC4R gene is amplified using theoligonucleotide pair SEQ ID NO: 8 and SEQ ID NO: 9, which yields a 226bp amplification product. The presence of a “T” residue results in anRFLP due to the creation of a TaiI restriction site. Thus, the presenceor absence of the SNP in the MC4R gene can be detected by restrictingthe amplification product with TaiI. In the absence of the SNP the 226bp is undigested, while in the presence of the SNP, the 226 bp isdigested to yield two fragments of 123 and 103 bp. The results of thedigestion reaction are analyzed using well-known DNA sizing techniquessuch as gel electrophoresis and the like. To aid in the detection of thedigestion products in cases where small amounts of DNA amplificationproducts are involved, the amplification products may be labeled with adetectable moiety to aid in the sensitivity of the detection methodsused. Such labeling tags and methods are known to those skilled in theart and it will be readily apparent whether such modifications would beneeded or desired.

Detection of the SNP present in the MC4R gene can also be convenientlyperformed by FRET analysis. Here the amplification product produced bythe oligonucleotide pair SEQ ID NO: 8 and SEQ ID NO: 9 is included in ahybridization reaction with oligonucleotide probes that serve ashybridization anchor and probe sequences. Conveniently, the anchor andprobe are labeled such that FRET analysis can be used to detect thepresence or absence of the SNP in the MC4R gene. The parametersaffecting hybridization and FRET analysis are well known to thoseskilled in the art. In addition, the foregoing are examples ofamplification products and hybridization probes that are suitable foruse with FRET analysis. It will be readily apparent to those skilled inthe art that modification may be made to the oligonucleotides that areused to synthesize the amplification products or probes while stillpermitting the practice of the present invention. As before, a varietyof amplification methods are suitable for use in the practice of thepresent invention and all such methods are intended to be within thescope of the invention.

The present invention also describes newly discovered single nucleotidepolymorphism sequences, previously not known in the art. In particular,the invention describes nucleic acids comprising a portion of the bovineCRH gene, further comprising a polymorphism at position 22 as defined bythe positions in SEQ ID NO: 1, and in which there is a “C” residue atposition 22. The invention also describes a nucleic acid comprising aportion of the bovine POMC gene, in which a T residue is present atposition 254 as defined by the positions in SEQ ID NO: 2.

Conveniently, purified and isolated nucleic acids comprising the SNPs ofthe invention may be recovered from animals by subjecting a sample ofgenomic DNA to an amplification procedure. Alternatively, it will bereadily apparent to those skilled in the art that DNA segmentscontaining the SNP could be artificially produced by oligonucleotidesynthesis technology, or by screening cDNA or genomic DNA librariesproduced from animals known to possess the polymorphisms.

Bovines, like all mammals, are diploid organisms possessing pairs ofhomologous chromosomes. Thus, at a typical genetic locus, an animal hasthree possible genotypes that can result from the combining of twodifferent alleles (e.g. A and B). The animal may be homozygous for oneor another allele, or heterozygous, possessing one of each of the twopossible alleles (e.g. AA, BB or AB).

The present invention provides a method of selecting individuallivestock animals based on the knowledge of an animal's CRH genotype.With respect to the SNP described in the present invention, the twopossible alleles are a “C” or “G” residue at position 22 as defined bySEQ ID NO: 1. The method of the invention comprises the steps ofdetermining the CRH alleles of an animal, such that it can be determinedwhether an animal is “CC”, “CG” or “GG” with respect to the CRH genelocus. The presence of a “G” allele is associated the desiredphenotypes. With the knowledge of the animal's genotype one can thenidentify and sort animals into groups of like phenotype, or otherwiseuse the knowledge of the genotype in order to predict which animals willhave the desired phenotypes of increased hot carcass weight, increasedend-of-test rib-eye area and increased adjusted weaning weight, ameasure of post-natal growth.

Here sorting can be taken to mean placing animals in physical groupingssuch as pens, so that animals of like genotype are kept separate fromanimals of a different genotype. This would be a useful practice in thecase of breeding programs where it would be desirable to produce animalsof particular genotypes. For example, it may be desirable to establishherds that are homozygous “GG” at the CRH gene, such that breeding amongthese animals would only produce more “GG” animals. Here keeping animalsof this genotype separate would be needed to ensure that “GG” animalsdid not have the opportunity to breed with animals possessing one ormore “C” alleles, which could result in the reproduction of animals witha reduced tendency to display the desired phenotypes associated with theCRH “G” allele. Furthermore, by ensuring that at least one animal in abreeding pair is “GG” at the CRH locus, conveniently allows for thefrequency of the “G” allele to be increased in the next, and subsequentgenerations.

Sorting may also be of a “virtual” nature, such that an animal'sgenotype is recorded either in a notebook or computer database. Here,animals could then be selected based on their known genotype without theneed for physical separation. This would allow one to select for animalsof desired phenotype where physical separation is not required.

The invention further provides a method of selecting individuallivestock animals based on the knowledge of an animal's POMC genotype.With respect to the POMC SNP described in the present invention, the twopossible alleles are a “C” or “T” residue at position 254 as defined bySEQ ID NO: 2. The method of the invention comprises the steps ofdetermining the POMC alleles of an animal, such that it can bedetermined whether an animal is “CC”, “CT” or “TT” with respect to thePOMC gene locus. With the knowledge of the animal's genotype one canthen sort animals into groups of like phenotype, or otherwise use theknowledge of the genotype in order to predict which animals will havethe desired phenotypes of increased shipping weight, increased averagedaily gain and increased hot carcass weight. The extent of thephenotypic response is directly related to the number of “T” alleles,such that an animal homozygous for the “T” allele, a “TT” animal, willdisplay the greatest phenotypic change for the desired phenotypes whencompared to animals with the “CC” genotype. Animals that are “CT”display an intermediate phenotypic response.

As described for the CRH gene, knowledge of the POMC genotype allows forthe selection and sorting of animals that will display desiredphenotypes. As before, sorting may be physical or virtual, and may beused in conjunction with animal breeding or other herd managementprograms.

The invention further comprises a method of selecting individuallivestock animals based on the knowledge of an animal's MC4R genotype.With respect to the MC4R SNP described in the present invention, the twopossible alleles are a “C” or “G” residue at position 1069 as defined bySEQ ID NO: 3. The method of the invention comprises the steps ofdetermining the MC4R alleles of an animal, such that it can bedetermined whether an animal is “CC”, “CG” or “GG” with respect to theMC4R gene locus. The presence of a “C” allele is associated the desiredphenotypes of increased hot carcass weight. With the knowledge of theanimal's genotype one can then sort animals into groups of likephenotype, or otherwise use the knowledge of the genotype in order topredict which animals will have the desired phenotypes of increased hotcarcass weight.

Knowledge of an animal's MC4R genotype provides a further advantage.Animals with one or two “C” alleles will display an increase in hotcarcass weight, regardless of the animal's CRH genotype. Thus, where thesole phenotype of interest is increased hot carcass weight, selectinganimals with either the “GG” CRH genotype or the “CC” or “CC” MC4Rgenotypes will accomplish the same goal, being the greatest increase inhot carcass weight. Any animals that have at least one “C” allele at theMC4R gene locus will display the increased hot carcass weight phenotype,and thus testing of animals for the presence of the CRH SNPs will not benecessary to detect and select the desired animals.

For the sub-group of animals that lack a “C” allele at MC4R, furthertesting for the presence of CRH SNP could detect additional animals thatwill display the desired phenotype. In any population where the “C” and“G” alleles for the MC4R gene are both present, testing for MC4Rgenotype provides the greatest chance of detecting animals that willdisplay the maximum increased hot carcass weight possible. This occursbecause two out of three possible combinations will have a “C” allele.In contrast, only one in three of the possible combinations, “GG”, inthe CRH SNP will exhibit the same maximum increase in hot carcassweight. The overall advantage will be that fewer animals will need to betested for both CRH and MC4R genotypes if the MC4R genotype of theanimal is determined first. Thus, based on the allele frequencies shownin Table V, it is more likely that an animal will have at least one “C”allele at MC4R than that it will be homozygous for the “G” allele atCRH.

As described for the CRH and POMC genes, knowledge of an animal's MC4Rgenotype allows for the selection and sorting of animals that willdisplay desired phenotypes. As before, sorting may be physical orvirtual, and may be used in conjunction with animal breeding or otherherd management programs.

It is a further aspect of the invention that animals can be selected asto their combined CRH and POMC genotypes. There is an advantage toselecting animals with a “GG” genotype at the CRH gene locus, and a “TT”genotype at the POMC gene locus, in that animals that are doublehomozygotes (“GG-TT” animals) will display the greatest phenotypicchange in the desirable phenotypes of increased hot carcass weight,increased shipping weight, increased average daily gain and increasedend-of-test rib-eye area, greater than that which would be obtained foranimals homozygous for only one of the CRH and POMC loci. Thus, throughthe method of the invention, animals with the most desirable combinationof growth-related phenotypes can be selected. As before, the selectionmay be physical or virtual, and the advantage of selecting animals basedon their combined CRH and POMC genotypes will be readily apparent tothose skilled in the areas of animal breeding, herd management and thelike.

In order to fully realize the utility of the invention, there are alsoprovided diagnostic kits that can be used to determine the CRH, POMC orMC4R genotypes of animals. In general, each of the kits comprisesoligonucleotide primers suitable to amplify the portions of each genecomprising the SNPs of the present invention. The kits comprise forwardand reverse primers suitable for amplification of a DNA sample takenfrom an animal. The sample may be from any tissue or fluid in whichgenomic DNA is present. Conveniently the sample may be taken from bloodskin or a hair bulb.

To for the presence of CRH SNP alleles, the kit comprises a forwardprimer comprising at it's 3′ end sequence identical to at least 10contiguous nucleotides within SEQ ID: 1, a reverse primer comprising atit's 3′ end a nucleotide sequence fully complementary to at least 10contiguous nucleotides with SEQ ID NO: 1. The primers are preferablyfrom 10 to 30 nucleotides in length, although variation in the length ofthe primer is not intended to be limiting. An example of suitableprimers would be those defined by SEQ ID NO: 4 and SEQ ID NO: 5.

In one embodiment of a diagnostic kit, the primers will be used toamplify DNA from a genomic DNA sample to produce amplification products,which can then be analyzed by restriction digest for the presence orabsence of the SNP at position 22 as defined by SEQ ID NO: 1. In analternative embodiment, the kit would further comprise hybridizationprobes adapted to distinguish between the two CRH alleles using FRETanalysis. In this embodiment, the use of FRET analysis is one suchmethod well known in the art that is suitable for detecting SNPs. Wherea method of analysis such as FRET is used, the hybridization probeswould be labeled with a detectable moiety to aid in the detection of theSNP. The types of detectable moieties suitable for use in FRET analysisare well known to those skilled in the art of molecular biology.

In another embodiment, where it is desired to test for the presence ofPOMC SNP alleles, the kit comprises a forward primer comprising at it's3′ end sequence identical to at least contiguous nucleotides within SEQID: 2, a reverse primer comprising at it's 3′ end a nucleotide sequencefully complementary to at least 10 contiguous nucleotides with SEQ IDNO: 2. The primers are preferably from 10 to 30 nucleotides in length,although variation in the length of the primer is not intended to belimiting. An example of suitable primers would be those defined by SEQID NO: 6 and SEQ ID NO: 7.

In one embodiment of a diagnostic kit, the primers will be used toamplify DNA from a genomic DNA sample to produce amplification products,which can then be analyzed by restriction digest for the presence orabsence of the SNP at position 254 as defined by SEQ ID NO: 2. In analternative embodiment, the kit would further comprise hybridizationanchor and detection probes adapted to distinguish between the two POMCalleles using FRET analysis. Where a method of analysis such as FRET isused, the hybridization probes would be labeled with a detectable moietyto aid in the detection of the SNP. The types of detectable moietiessuitable for use in FRET analysis are well known to those skilled in theart of molecular biology.

In yet another embodiment, where it is desired to test for the presenceof MC4R SNP alleles, the kit comprises a forward primer comprising atit's 3′ end sequence identical to at least 10 contiguous nucleotideswithin SEQ ID: 3, a reverse primer comprising at it's 3′ end anucleotide sequence fully complementary to at least 10 contiguousnucleotides with SEQ ID NO: 3. The primers are preferably from 10 to 30nucleotides in length, although variation in the length of the primer isnot intended to be limiting. An example of suitable primers would bethose defined by SEQ ID NO: 8 and SEQ ID NO: 9.

In one embodiment of a diagnostic kit, the primers will be used toamplify DNA from a genomic DNA sample to produce amplification products,which can then be analyzed by restriction digest for the presence orabsence of the SNP at position 1069 as defined by SEQ ID NO: 3. In analternative embodiment, the kit would further comprise hybridizationprobes adapted to distinguish between the two MC4R alleles by FRETanalysis. In this embodiment, the use of FRET analysis is one suchmethod well known in the art that is suitable for detecting SNPs. Wherea method of analysis such as FRET is used, the hybridization probeswould be labeled with a detectable moiety to aid in the detection of theSNP. The types of detectable moieties suitable for use in FRET analysisare well known to those skilled in the art of molecular biology.

It will also be obvious to one skilled in the art that diagnostic kitswill include additional reagents including, but not limited to lysingbuffers for lysing cells contained in a sample, dNTP's, reaction buffer,an amplifying enzyme and combinations thereof. Kits may also includeaccessory diagnostic agents such as the restriction enzyme used todetect the SNP, or detection reagents to reveal the presence ofdetectable moieties. For example, it is well known in the art, andespecially where only limited quantities of DNA are available, to usesensitive detection techniques such as Southern blot hybridization,chromatography or mass spectroscopy in order to detect specificamplification products, or specific restriction digest products derivedfrom amplification products as derived herein. Thus, the precise meansof detecting the various SNPs from the amplification or restrictiondigest products could be performed by a variety of techniques well knownto those skilled in the art of molecular biology, and the presentinvention is intended to encompass those methods of detection.

The diagnostic kits as referred to herein may be individually packagedfor each individual gene locus to be tested, or the required reagentsrequired to test for polymorphisms in all three genes could be presentin a single kit. Such variation is common in diagnostic kits and it notintended to be limiting of the invention.

EXAMPLES

In terms of demonstrating the practice of the inventions the followingexamples are provided.

Materials and Methods: Cattle:

A group of 256 tan-colored Charolais-cross steers were divided into twogroups of 128 animals each, and either limit-fed a grain diet orfull-fed a forage-based diet during the backgrounding phase (90 dayperiod). During the finishing phase each of these two groups weredivided into half and allowed a full or limited high grain diet; thiswas based on voluntary intake. The full diets were 100% ad libitum andthe limited diets were 95% ad libitum. Live animal weight, ultrasoundmeasurements of rib-eye area (REA) and average daily gain (ADG) datawere collected. Steers were sent for slaughter as they approached atarget shipping weight of 635 kg (mean=636.6 kg, SD=21.48). They wereweighed every two weeks then on two consecutive days and transported forslaughter within a week where hot carcass weight (HCW) data wascollected. Adjusted weaning weights were available in a second group ofanimals (n=255) that included the Canadian Beef Reference Herd (n=132;Schmutz et al., 2001) and 123 animals from three ranches.

SNP Identification in CRH Gene:

Exon 2 of the CRH gene was amplified and sequenced as described inBuchanan et al. (2002b; GenBank accession number AF340152). Briefly, thefollowing single-stranded DNA primers were used to amplify a 254 bpfragment:

Forward: 5′- ATGCGACTGCCGCTGCTCG -3′ Reverse: 5′- AGAGAGGGGAGCAGCCCG -3′

The 20 ul reaction contained: 100 ng of DNA, 10×PCR buffer, 5×Q-solution (Qiagen), 0.2 mM dNTPs, 0.5 units Taq polymerase, 4 pmolforward primer, and 4 pmol reverse primer. The amplification programconsisted of one cycle of 95° C. for 2 minutes, followed by 35 cyclesof: denaturation at 95° C. for 45 sec, annealing at 55° C. for 30 secand extension at 72° C. for 45 sec, followed by a final cycle ofextension at 72° C. for 3 minutes. Sequencing of PCR products wasperformed on an AB1373 sequencer (Applied Biosystems). Sequence wasaligned and compared for variation using Sequencher 4.1.2 (Gene CodesCorporation).

PCR-RFLP Analysis: CRH4

The CRH4 SNP is defined by a C to G transition at position 22 of SEQ IDNO: 1. Wherever the term “CRH4” or “CR14 SNP” is used it is meant torefer to the nucleotide present at position 22 of SEQ ID NO: 1. CRH4genotypes were derived from the amplification and subsequent digestionof the product with DdeI (New England BioLabs). The following primerswere used to amplify a 129 bp product:

Forward: 5′- GCGCCCGCTAAAATGCGACTGA -3′ Reverse: 5′-CTGTGATGCCTGCCGGGCAC -3′

The 25 ul amplification reaction contained 50 ng of genomic bovine DNA,0.2 uM of each primer, 0.2 mM dNTPs, 45 mM Tris-HCl pH 8.8, 11 mM(NH₄)₂SO₄, 4.5 mM MgC₂, 6.7 mM β-mercaptoethmol, 4.5 mM EDTA, 0.25 mMspermidine, 10% DMSO and 0.65 U Taq DNA polymerase (Invitrogen). Thecycling protocol was 2 min at 94° C., 35 cycles of 94° C. for 1 min, 62°C. for 45 sec, 72° C. for 50 sec, with a final extension at 72° C. for 4min. A 2-hour digestion with DdeI was carried out in a 37° C. waterbath. The digested PCR-products were separated on a 4% agarose gel.Digestion of the 129 bp PCR product with DdeI produced fragments of 88and 41 bp, due to the presence of a DdeI recognition site at nt 87 ofthe PCR product. This site was present in the DNA of all animals tested.The CRH4 SNP introduces a second DdeI recognition site due the presenceof a G residue at nucleotide 87, resulting in further digestion of the88 bp fragment further into fragments of 69 and 19 bp upon digestionwith DdeI.

POMC, LEP and MC4R Genotyping:

Genotyping methods for SNPs present in POMC, MC4R and LEP havepreviously been described (Buchanan et al., 2002a; 2002b; Thue et al.,2003).

Statistical Analysis:

A regression analysis was carried out in the group of 255 cattle todetermine if the number of copies of a CRH allele significantly affectedadjusted weaning weight. In the steers (fed two different backgroundingrations and finishing diets) statistical analyses were performed usingthe general linear model (GLM) procedure of SAS (SAS, 1998). Nosignificant effects were observed based on backgrounding (forage vs.grain) or finishing (limited vs. ad libitum) diets or the interactionbetween the two and hence these were deleted from the model. Nosignificant effects were observed with leptin or between the four orthree gene interactions and hence these were deleted from the model.

In steers, the model used to determine the effects of genotype on ADG,EOT-REA, shipping weight and HCW was:

Y_(kmno)=μ+GCRH_(k)+GPOMC_(m)+GMC4R_(n)+GCRH×GPOMC_(km)+GCRH×GMC4R_(kn)+GPOMC×GMC4R_(mn)+e _(kmno)

Where: Y_(kmno) is an observation of the dependent variable (end-of-testREA, Shipping weight, hot carcass weight (HCW) and average daily gain(ADG)); μ is the overall population mean for the variable; GCRH_(k) isthe effect of the kth genotype of CRH4 (k=1 (GC), 2 (CC), 3 (GG));GPOMC_(m) is the effect of the mth genotype of POMC (m=1 (CC), 2 (CT), 3(TT)); GMC4R is the effect of the nth genotype of MC4R (n=1 (GG), 2(GC), 3 (CC)); GCRH×GPOMC_(km), GCRH×GMC4R_(kn), and GPOMC×GMC4R_(mn)are effects of the gene interaction and e_(kmno) is the random errorassociated with the observation. Treatments comparisons used leastsquares means by PDIFF options (SAS, 1998). Significance was declared atP<0.05.

Results:

The CHR4, POMC, MC4R and LEP genes were genotyped in 256 steers and thenanalyzed using GLM for association with average daily gain (ADG),end-of-test rib-eye area (EOT-REA), shipping weight and hot carcassweight (HCW). The CRH4 SNP was positively correlated with EOT-REA andHCW (Table I). The POMC SNP was positively correlated with shippingweight and HCW and there was evidence of a trend associated with ADG(Table I). MC4R showed a trend with HCW (Table I).

Interactions between two sets of genes were observed in the case of twotraits. The CRH and POMC genes appeared to interact with respect toEOT-REA (P=0.0407), while the CRH and MCR4 genes appeared to interactwith respect to HCW (P=0.02) (Table 1).

In the case of the interaction between the CRH and MCR4 genes, HCWincreased by 26 kg where animals were homozygous at CRH for the “C”allele, and had either one or two MC4R “C” alleles (i.e. “CC” or “CG” atthe MCR4 SNP site) as compared to animals with an MC4R “GG” genotype(P=0.008 and P=0.008). However, these same animals (CC-CC or CC-CG atMCR4) were not significantly different from animals that had one or two“G” alleles at CRH.

The data lead to the conclusion that, if an animal has either the “GG”genotype at the CRH gene locus, or, at least one “C” allele at the MC4Rgene locus, then they will display the desired phenotype of increase hotcarcass weight. Thus, CRH and MC4R operate as parallel switches withrespect to the increased hot carcass weight phenotype such that if oneswitch is on, the position of the other is irrelevant.

Carcass yield least square means for CRH and POMC genotypes are reportedin Tables II and III respectively. The presence of a “G” allele at CRH4is associated with an increase in weight. In the case of POMC, the “T”allele is associated with increased weights. Furthermore, the effect ofa “T” allele appears to be additive with each copy of the alleleresulting in an increase of 9 kg. The “C” allele at MC4R is associatedwith an increase in HCW (LS Means CC=378, CG=377 and GG=368 SEM=1.79),and acts in a dominant fashion.

The expected gains in cm² of rib-eye area or live and carcass weight inkilograms are shown in Table IV. Allele frequencies observed in the 256steers for CRH4, POMC and MC4R are shown in Table V. The allelefrequencies were such that adequate representation of all genotypeswould have been present in the test population.

Three SNPs have now been identified in the CRH gene, two in thepro-peptide region and another in the signal sequence. The C/G SNP atcodon 4 of CRH (position 22 of SEQ ID NO: 1) appears to be the mutationassociated with effects on adjusted weaning weight, a measure ofpost-natal growth and carcass weight. A Pro within the CRH signalsequence at codon 4 has been previously reported in both sheep (Roche etal., 1988) and humans (Shibabara et al., 1983). Similarly, in theGenbank database a Pro at this same site has been reported in anotherovine sequence, two porcine, and two more human CRH sequences (AccessionNumbers J00803, AF440229, Y15159, NM000756 and BC011031 respectively).

The introduction of an Arg at position 4 in the signal sequence couldlead to a reduction in the levels of circulating CRH (FIG. 3), resultingin a decrease in the growth inhibition effect normally associated withthis hormone.

The data indicate that the CRH4 SNP is predictive of increased EOT-REAand HCW. The data from animals that were “GG-TT” homozygotes at CRH andPOMC respectively, suggests that the presence of the CRH4 allele doesnot result in an increase in shipping weight above that observed forPOMC “TT” homozygotes alone. The data from animals with various CRH andMC4R genotypes showed that the greatest increase in hot carcass weightcould be achieved either by having CRH animals homozygous for the “G”allele SNP, or animals with at least one “C” allele at the MC4R locus.

We had previously sequenced POMC as a candidate gene for ADG and carcassweight. We mapped it directly under QTL peaks (Thue et al., 2003) andthe current results confirm its association with ADG (P=0.07) andcarcass weight. Whatever the molecular mechanism through which POMCpolymorphisms act, the associations with increased ADG, shipping and hotcarcass weight can be capitalized upon by selecting cattle that arehomozygous for the “T” allele at POMC. The effect seen on shippingweight is likely to be an under estimate, based on how these animalswere shipped (target weight) as compared to the usual practice. Theusual practice in feedlots is to either use a set number of days on feedor visually appraise the weight of an animal, and then actually weigh atruckload of cattle.

Since CRH affects POMC levels both directly in the hypothalamus, andindirectly by affecting the release of glucocorticoids, it was importantto determine if the associations between these genes and the varioustraits we have examined were dependent or independent effects of theSNPs that have been studied. As there was no interaction effect observedwith respect to HCW, the data are consistent with the premise that thesegenes act independently of each other. As a result, testing for thepresence of either the CRH or POMC SNPs will be useful as a predictor ofHCW. In addition, given that the allele frequencies of each of thealleles associated with the most desirable traits (increase weights) arefairly common (0.37 for the CRH4 “G” allele, and 0.23 for the POMC “T”allele), it will be possible to use the genetic tests of the presentinvention to select for animals that are homozygous for both the CRH4 Gand POMC “T” alleles. Selecting for such “GG-TT” animals will allow forthe concurrent improvement REA, shipping weight, HCW and ADG. Finally,the association observed between adjusted weaning weight and the CRH4SNP means the cow-calf producer will benefit from genetic selectionmethods based on this SNP.

TABLE I General Linear Model Probability values for gene(s) with TraitAssociations. Gene or Gene EOT- interaction ADG REA Shipping HCW CRH40.37 0.034* 0.35 0.0015** POMC 0.07 0.45 0.0078** 0.006** MC4R 0.88 0.780.60 0.085 CRH4 × POMC 0.89 0.047* 0.63 0.17 CRH4 × MC4R 0.28 0.46 0.210.02* *= P < 0.05; **= P < 0.01

TABLE II Effects of CRH4 on beef cattle performance (LS means) CRH4Trait CC GC GG SEM* EOT-REA 88.6b 92.7ab 94.6a 0.84 Shipping Wt 631.5638.5 641.5 2.55 HCW 367.5c 373.7bc 381. 8a 1.64 *SEM = pooled standarderror of mean; Means with different letters in the same row aresignificantly different (P < 0.05)

TABLE III Effects of POMC on beef cattle performance (LS means) POMCTrait CC CT TT SEM* ADG 1.62 1.69 1.72 0.028 EOT-REA 92.5 93.3 90.2 1.11Shipping Wt 628.0b 637.4a 646.0a 3.37 HCW 368.8b 376.2a 378.0a 2.16 *SEM= pooled standard error of mean; Means with different letters in thesame row are significantly different (P < 0.05)

TABLE IV Increase in REA and pounds expected from selecting animals GGat CRH4 or TT at POMC or both GG and TT. Trait CRH4 POMC CRH4 + POMC EOTREA 6 cm² — 6 cm² Ship weight  18 kg 18 kg Hot carcass weight 14.3 kg9.2 kg 23.5 kg

TABLE V Allele frequencies for CRH, POMC and MC4R CRH4 POMC MC4R G =0.37 T = 0.23 C = 0.66 C = 0.63 C = 0.77 G = 0.34

TABLE VI Summary of the Phenotypes Associated with the CRH, POMC andMC4R SNPs. Gene SNP Phenotype(s) CRH C-G transition HCW, EOTREA, AWWPOMC C-T transition ADG, HCW, SW MC4R G-C transition HCW The followingabbreviations are used in the tables: HCW—hot carcass weight; EOTREA—endof test rib eye area; AWW—adjusted weaning weight; ADG—average dailygain; SW—shipping weight.

The foregoing is considered as illustrative only of the principles ofthe invention. Further, since numerous changes and modifications willreadily occur to those skilled in the art, it is not desired to limitthe invention to the exact construction and operation shown anddescribed, and accordingly, all such suitable changes or modificationsin structure or operation which may be resorted to are intended to fallwithin the scope of the claimed invention.

SEQUENCE LISTING Number of SEQ ID NOS: 9 SEQ ID NO: 1

LENGTH: 584 base pairs

TYPE: DNA

ORGANISM: Bos taurusFEATURE: SNP's present at nucleotides 22 (“CRH4”), 145 (“CRH45”) and 240(“CRH77”).

OTHER INFORMATION: GenBank Accession AF340152 SEQUENCE:

  1 cgcccgctaa aatgcgactg ccgctgctcg tgtccgtggg cgtcctgctg gtggctctgc 61 tgccctcccc gccatgcagg gccctcctca gccgggggcc catcccgggt gcccggcagg121 catcacagca cccccagccc ctgagtttct tccagccgcc gccgcagccc caggaacccc181 aggctctgcc caccctactc cgtgttgggg aggaatactt cctccgcctg ggtaacctcg241 atgagacccg ggctgctccs ctctctcccg ccgcctcgcc tctcgccagc agaagcagca301 gtcgcctttc tccggacaag gtggccgcca actttttccg agcgctgctg cagccccggc361 gcccattcga cagcccagcg ggtcccgcgg aacgcggcac ggagaacgcc ctcggcagcc421 gccaggaggc gccggccgcc aggaagaggc gatcccagga acctcccatc tccctggatc481 tcaccttcca cctcctccga gaagtcttgg aaatgaccaa ggccgatcag ttagcacagc541 aagctcatar caayaggaaa ctgttggaca ttgctgggaa atga

SEQ ID NO: 2

LENGTH: 1002 base pairs

TYPE: DNA

ORGANISM: Bos taurusFEATURE: SNP at position 254

OTHER INFORMATION: GenBank Accession J00021 SEQUENCE:

  1 gcggagggag tggaaggctc aggcggcgcg cttgaggggc gggtgaacgc cgcggcctgg 61 agtgggcggg gcctgacgcg ctctgccgct ctccgcaggc gtgcatccgg gcctgcaagc121 ccgacctctc cgccgagacg ccggtgttcc ccggcaacgg cgatgagcag ccgctgactg181 agaacccccg gaagtacgtc atgggccatt tccgctggga ccgcttcggc cgtcggaatg241 gtagcagcag cagcggagtt gggggcgcgg cccagaagcg cgaggaggaa gtggcggtgg301 gcgaaggccc cgggccccgc ggcgatgacg ccgagacggg tccgcgcgag gacaagcgtt361 cttactccat ggaacacttc cgctggggca agccggtggg caagaagcgg cgcccggtga421 aggtgtaccc caacggcgcc gaggacgagt cggcccaggc ctttcccctc gaattcaaga481 gggagctgac cggggagagg ctcgagcagg cgcgcggccc cgaggcccag gctgagagtg541 cggccgcccg ggctgagctg gagtatggcc tggtggcgga ggcggaggct gaggcggccg601 agaagaagga ctcggggccc tataagatgg aacacttccg ctggggcagc ccgcccaagg661 acaagcgcta cggcgggttc atgacctccg agaagagcca aacgcccctt gtcacgctgt721 tcaaaaacgc catcatcaag aacgcccaca agaagggcca gtgagggcgc agcgggcagg781 ggcctctctc cgcggaaagt tgaccctgaa ggcctctctt ctgccctcct accgcctcgc841 agcctgggtg aggattcgcc caggcagtga tggcgccagg tatcccgact cttaaagctg901 tctgtagtta agaaataaaa cctttcaagt ttcacgaata ttgactgggt gaattaaaaa961 cgcatttcca tcaagtaaag ggcagtacat attggagggg cg

SEQ ID NO: 3

LENGTH: 1809 base pairs

TYPE: DNA

ORGANISM: Bos taurusFEATURE: SNP at position 1069OTHER INFORMATION: Genbank Accession No. AF265221

SEQUENCE:

   1 cagcctaaga tttccaagtg atgctgacca gagccacact tgaaagagac tgaaaacttc  61 ctttccagct ccggagcatg ggacatttat tcacagcagg catgccactc tccgccgcct 121 aactttcgtt tggggcaagt caagactgga gaaaggtgct gaggctgcca gatccaggag 181 gttcagtcag tccagagggg acctgaatcc aaaatgaact ctacccagcc ccttgggatg 241 cacacctctc tccactcctg gaaccgcagc gcccacggaa tgcccaccaa tgtcagtgag 301 tccctggcaa aaggctactc ggacgggggg tgctatgagc agctctttgt ctctcccgag 361 gtgtttgtga ctctgggggt catcagcttg ttggagaata ttctggtgat cgtggccata 421 gccaagaaca agaatctgca ctcacccatg tactttttca tctgcagcct ggctgtggct 481 gacatgttgg tgagcgtttc caacgggtcg gaaaccattg tcatcaccct gctgaacagc 541 acggacacgg acgcgcagag cttcacggtg gatattgaca atgtcattga ctcggtgatc 601 tgtagctcct tgcttgcctc catctgcagc ttgctgtcga tcgcggtgga caggtacttc 661 actatcttct atgcgctcca gtaccataac atcatgacgg tgaagcgggt ggcgatcacc 721 atcagcgcca tctgggcagc ctgcacggtg tcgggcgtct tgttcatcat ttactcagac 781 agcagtgctg ttatcatctg cctcatcacc gtgttcttca ccatgctggc tctcatggcg 841 tctctctatg tccacatgtt cctcatggcc agactccaca ttaagaggat cgcggtcctg 901 ccaggtagcg gcaccatccg ccagggcgcc aacatgaagg gggcgattac cctgaccata 961 ctgatcgggg tctttgttgt ctgctgggcc cccttcttcc tgcacctgat attctacatc1021 tcttgtcccc agaacccata ctgtgtgtgt ttcatgtctc actttaacct gtacctcatc1081 ctcatcatgt gcaattccat cattgaccct ctgatttatg ccctgcggag ccaagaactg1141 aggaaaacct tcaaagagat catttgttgc tctcctctag gtggcctctg tgatttgtct1201 agcagatatt aaatggggac aaacgcgatg ctaaacacaa gcttaagaga ctttctcctt1261 ctcatatgta caacctgaac agtctgtatc agccacagct ttttcttctg tgtagggcat1321 ggagtgaaaa tttctattgt atcagttgaa gtttgtgatt tttttctgat gtgaaacagt1381 gcccagtctt ggtgtatttt taatgtcatg ctactttctg gctgtaaaat gtgaatccac1441 atcacaggtt ataggcacta tgcatttata aaaaaagaag aaaaaaagtc cttatgagga1501 gtttaacagt gtttccttct tgttatttac aaggatgtga cactttgctt gcttttgtaa1561 catggaaatc acagcttcat taagtatatc ctcataagtg gtttttttat gttatacttt1621 acaacactga agtgtaaaaa tttgattcta gcatttaggg gagaaatatt gagaacatat1681 tgcttaatca taaaaaacaa gctgaaattt caggtaattt aataagactt tctcattcat1741 tcttcctgtg cagaagttga aatgaagctt gtattgggag aaaaacagtt acttaaaaaa1801 aaaaaaaaa

SEQ ID NO: 4 LENGTH: 22 TYPE: DNA

ORGANISM: Bos taurus

FEATURE:

OTHER INFORMATION: Forward primer for DNA amplification of sequenceswithin SEQ ID NO: 1.

SEQUENCE:

1 gcgcccgcta aaatgcgact ga 22

SEQ ID NO: 5 LENGTH: 20 TYPE: DNA

ORGANISM: Bos taurus

FEATURE:

OTHER INFORMATION: Reverse primer for DNA amplification; sequence is thereverse complement of the corresponding sequence in SEQ ID NO: 1.

SEQUENCE:

1 ctgtgatgcc tgccgggcac 20

SEQ ID NO: 6 LENGTH: 21 TYPE: DNA

ORGANISM: Bos taunis

FEATURE:

OTHER INFORMATION: Forward primer for DNA amplification of sequenceswithin SEQ ID NO: 2.

SEQUENCE:

1 cgtgcatccg ggcctgcaag c 21

SEQ ID NO: 7 LENGTH: 23 TYPE: DNA

ORGANISM: Bos taunts

FEATURE:

OTHER INFORMATION: Reverse primer for DNA amplification; sequence is thereverse complement of the corresponding sequence in SEQ ID NO: 2.

SEQUENCE:

1 gtcagctccc tcttgaattc gag 23

SEQ ID NO: 8 LENGTH: 20 TYPE: DNA

ORGANISM: Bos taurus

FEATURE:

OTHER INFORMATION: Forward primer for DNA amplification of sequenceswithin SEQ ID NO: 3.

SEQUENCE:

1 taccctgaccatactgatcg

SEQ ID NO: 9 LENGTH: 22 TYPE: DNA

ORGANISM: Bos taurus

FEATURE:

OTHER INFORMATION: Reverse primer for DNA amplification; sequence is thereverse complement of corresponding sequence in SEQ ID NO: 3.

SEQUENCE:

1 xxxxxxxxxx xxxxxxxxxx 20

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D.,    Vage D-I., Van de Weghe A., Varvio S., Velmala R., Vilkld J.,    Weikard, R., Woodside C., Womack J. E., Zanotti M., Zaragoza P., A    medium-density genetic linkage map of the bovine genome, Mammal.    Genome 8 (1997) 21-28.-   3. Buchanan F. C., Thue T. D., Winkelman-Sim D. C., Plante Y.,    Schmutz S. M., Two QTLs for growth map to bovine chromosome 14,    27^(th) International Conference on Animal Genetics, Jul. 22-26    2000, Minneapolis, Minn.-   4. Buchanan F. C., Fitzsimmon C. J., Van Kessel A. G., Thue T. D.,    Winkelman-Sim D. C., Schmutz S. M., Association of a missense    mutation in the bovine leptin gene with carcass fat content and    leptin mRNA levels, Genet. Sel. Evol. 34 (2002) 105-116.-   5. Buchanan F. C., Thue T. D., Elsaesser E. D., Winkelman-Sim D. C.,    A corticotrophin-releasing hormone polymorphism associated with    post-natal growth in Beef cattle, Proceedings of the 7^(th) World    Congress on Genetics Applied to Livestock Production. 2002 Paper.    CD-ROM communication n° 11-32.-   6. Dunn A. J., Berridge, C. W., Physiological and behavioral    responses to corticotropin-releasing factor administration: is CRF a    mediator of anxiety or stress responses? Brain Research Reviews    15 (1990) 71-100.-   7. Grobet L., Poncelet D., Royo LJ., Brouwers B., Pirotiin D.,    Michaux C., Menissier F., Zanotti M., Dunner S., Georges, M.,    Molecular definition of an allelic series of mutations disrupting    the myostatin function and causing double-muscling in cattle,    Mammal. Genome 9 (1998) 210-213.-   8. Houseknecht K L., Baile C. Z., Matteri R. L., Spurlock M C., The    biology of leptin: A review, J. Anim. Sci. 76 (1998) 1405-1420.-   9. Kress D. D., Burfening P. J., Miller P. D., Vaniman D., Beef sire    expected progeny differences calculated by three mthods, J. Anim.    Sci. 44 (1977) 195-202.-   10. Liu H-X., Chew S. L., Cartegni L, Zhang M. Q., Krainer A. R.,    Exonic splicing enhancer motif recognized by human SC35 under    splicing conditions, Molecular and cellular biology 20    (2000)1063-1071.-   11. Liu H-X., Cartegni L., Thmag M. Q., Krainer A. R., A mechanism    for exon slipping caused by nonsense or missense mutations in BRCA1    and other genes, Nat. Genet. 27 (2001) 55-58.-   12. Marsh D. J., Hollopeter G., Huszar D., Laufer R., Yagaloff K A.,    Fisher S. L. Burn P. Palmiter R. D., Response of melanocortin-4    receptor-deficient mice to anorectic and orexigenic peptides, Nat.    Genet. 21 (1999)119-122.-   13. Pritchard L. E., Tumbull A. V., White A., Pro-opiomelanocortin    processing in the hypothalamus: impact on melanocortin signalling    and obesity, J. Endocrinol. 172 (2002) 411-421.-   14. Roche P. J., Crawford R. J., Fernley R. T., Tregear G. W.,    Coghlan, J. P., Nucleotide sequence of the gene coding for ovine    corticotropin-releasing factor and regulation of its mRNA levels by    glucocorticoids, Gene 71 (1988) 421-431.-   15. SAS (1998) SAS/STAT User's Guide (Release 8.02). SAS Inst. Inc.,    Cary, N. C.-   16. Schmutz S. M., Buchanan F. C., Winkelman-Sim D. C., Pawlyshyn    V., Plante Y., McKinnon J. J., Fournier, B. P., Development of the    Canadian Beef Reference Herd for gene mapping studies,    Theriogenology 55 (2001) 963-972.-   17. Schumann F. J., Janzen E. D., McKinnon, J. J., Prophylactic    tilmicosin medication of feedlot calves at arrival, Can. Vet. J.    31 (1990) 285-288.-   18. Sharpe P. M., Haynes N. B., Buttery P. J., Glucocorticoid status    in growth, in: Buttery P. J., Haynes N. B., Lindsay D. B. (Eds.),    Control and Manipulation of Animal Growth, Butterworths, London,    1986, p 207-222.-   19. 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1. A method for distinguishing bovines having a MC4R gene polymorphism,comprising: isolating genomic DNA from a bovine; amplifying a region ofthe bovine MC4R gene using an oligonucleotide pair to form nucleic acidamplification sequences comprising amplified MC4R gene polymorphismsequences; detecting a polymorphism present in the MC4R gene at position1069 of SEQ ID NO: 3; analyzing the polymorphism, and wherein thepresence of a “G” residue indicates the presence of the MC4Rpolymorphism, as compared to bovines with a “C” residue at position 1069of SEQ ID NO:
 3. 2. The method of claim 1 wherein the oligonucleotidepair comprises SEQ ID NO: 8 and SEQ ID NO:
 9. 3. The method of claim 1wherein the polymorphism detected is a restriction fragment lengthpolymorphism (RFLP).
 4. The method of claim 3 wherein the RFLP is thepresence or absence of a TaiI restriction site in a nucleic acidamplification product produced by amplification of the MC4R gene.
 5. Themethod of claim 1 further comprising the inclusion of a detectablemoiety such that the amplification product comprises a labeledamplification product.
 6. The method of claim 5 wherein the detectablemoiety is selected from the group consisting of fluorescent,bioluminescent, chemiluminescent, radioactive and colorigenic moieties.7. The method of claim 1 further comprising: contacting the nucleic acidamplification product with a hybridization probe; wherein thehybridization probe comprises at least one oligonucleotide labeled witha detectable moiety; under suitable conditions permitting hybridizationof the at least one oligonucleotide to the amplification product to forma hybridization complex; and wherein the presence of the detectablemoiety in the hybridization complex indicates the presence of a MC4Rpolymorphism.
 8. The method of claim 1 wherein the nucleic acidamplification product is produced by an amplification method selectedfrom the group of polymerase chain reaction (PCR), strand displacementamplification (SDA), nucleic acid sequence based amplification (NASBA),rolling circle amplification, T7 polymerase mediated amplification, T3polymerase mediated amplification and SP6 polymerase mediatedamplification.
 9. An isolated and purified nucleic acid comprising aportion of the bovine CRH gene, further comprising a polymorphism atposition 22 as defined by the positions in SEQ ID NO: 1, and in whichthere is a “C” residue at position
 22. 10. An isolated and purifiednucleic acid comprising a portion of the bovine POMC gene, furthercomprising a polymorphism at position 254 as defined by the positions inSEQ ID NO: 2, and in which there is a “T” residue at position
 254. 11. Amethod of selecting individual livestock animals based on the knowledgeof an animal's MC4R genotype, comprising the steps of: determining theMC4R alleles of an animal; wherein the alleles of an animal will be oneof “CC”, “CG”, or “GG” at position 1069 of SEQ ID NO: 3; and wherein a“CG” or “GG” genotype is associated with increased hot carcass weight.12. The method of claim 11, wherein the only phenotype of interest ismaximum increased hot carcass weight, and wherein an animal is firsttested to determine the animal's MC4R genotype; and wherein, if theanimal is homozygous for the “G” allele at the MC4R gene locus theanimal is then tested to determine its CRH genotype, such that an animalthat is homozygous for the “G” allele at the CRH gene locus will displaythe desired phenotype of maximum increased hot carcass weight.
 13. Adiagnostic kit for determining the CRH genotype of a bovine animal, thekit comprising: oligonucleotide primers for amplifying a portion of theCRH gene; the primers comprising a forward primer comprising at its 3′end sequence identical to at least 10 contiguous nucleotides within SEQID: 1; a reverse primer comprising at its 3′ end a nucleotide sequencefully complementary to at least 10 contiguous nucleotides with SEQ IDNO: 1; and wherein the forward and reverse primers are from 10 to 30nucleotides in length and in a PCR amplification reaction will produce anucleic acid product amplification product containing a residuecorresponding to position 2 of SEQ ID NO:
 1. 14. The kit of claim 13wherein the primers comprise the oligonucleotides SEQ ID NO: 4 and SEQID NO:
 5. 15. The kit of claim 13 wherein the primers are labeled with adetectable moiety.
 16. The kit of claim 13 further comprising at leastone oligonucleotide, labeled with a detectable moiety and suitable foruse as a hybridization probe.
 17. A diagnostic kit for determining thePOMC genotype of a bovine animal, the kit comprising: oligonucleotideprimers for amplifying the POMC gene; the primers comprising a forwardprimer comprising at its 3′ end sequence identical to at least 10contiguous nucleotides with SEQ ID: 2; a reverse primer comprising atits 3′ end a nucleotide sequence fully complementary to at least 10contiguous nucleotides with SEQ ID NO: 2; at least one additionalreagent selected from the group consisting of a lysing buffer for lysingcells contained in a sample, dNTP's, reaction buffer, an amplifyingenzyme and a combination thereof; and wherein the forward and reverseprimers are from 10 to 30 nucleotides in length and in a PCRamplification reaction will produce a nucleic acid product amplificationproduct containing a residue corresponding to position 254 of SEQ ID NO:2.
 18. The kit of claim 17 wherein the primers comprise theoligonucleotides SEQ ID NO: 6 and SEQ ID NO:
 7. 19. The kit of claim 17wherein the primers are labeled with a detectable moiety.
 20. The kit ofclaim 17 further comprising an oligonucleotide, labeled with adetectable moiety and suitable as a hybridization probe.
 21. A kit fordetermining the MC4R genotype of an animal, the kit comprising:oligonucleotide primers for amplifying the MC4R gene; the primerscomprising a forward primer comprising at its 3′ end sequence identicalto at least 10 contiguous nucleotides with SEQ ID: 3; a reverse primercomprising at its 3′ end a nucleotide sequence fully complementary to atleast 10 contiguous nucleotides with SEQ ID NO: 3; at least oneadditional reagent selected from the group consisting of a lysing bufferfor lysing cells contained in a sample, dNTP's, reaction buffer, anamplifying enzyme and a combination thereof; and wherein the forward andreverse primers are from 10 to 30 nucleotides in length and in a PCRamplification reaction will produce a nucleic acid product amplificationproduct containing a residue corresponding to position 1069 of SEQ IDNO:
 3. 22. The kit of claim 21 wherein the primers comprise theoligonucleotides SEQ ID NO: 8 and SEQ ID NO:
 9. 23. The kit of claim 21wherein the primers are labeled with a detectable moiety.
 24. The kit ofclaim 21 further comprising at least one oligonucleotide, labeled with adetectable moiety and suitable or use as a hybridization probe.